Human granulocytic anaplasmosis (HGA) is an emerging zoonosis, and one of the most prevalent life- threatening tick-borneillnesses in North America. This disease is caused by infection with the obligatory intracellular bacterium, Anaplasma phagocytophilum. Given the propensity of A. phagocytophilum to cause severe and sometimes deadly diseases, its increasing prevalence throughout the world, and limited treatment choices and preventive measures available, there is a critical need to understand this pathogen and its pathogenesis. Although it is known that cholesterol is essential for this bacterium, and cholesterol is a critical determinant of HGA pathogenesis, how this bacterium acquires cholesterol is unknown. Our long-term goal is to understand how Anaplasma acquires cholesterol from host cells and apply this knowledge to prevent and treat severe HGA. The objective here is to determine the path by which cholesterol in serum low-density lipoprotein (LDL) taken up by host cells traffics from late endosomes to Anaplasma inclusions, which may reveal a novel target for intervention. Our central hypothesis is that Anaplasma modulates the normal LDL- derived cholesterol (LDL-CHOL) intracellular traffic at a critical step in order to appropriate cholesterol. To test this hypothesis, Specific aim 1 is to determine the mechanism by which LDL-CHOL is delivered to Anaplasma inclusions. Our working hypothesis is that Anaplasma infection up-regulates a subset of Niemann-Pick type C- 1 (NPC1) vesicles containing LDL-CHOL, but not lysosomal markers, which traffics to the Anaplasma inclusions; NPC1 function is required for LDL-CHOL delivery to bacteria, thus promoting infection. To test the working hypothesis, our approach is to characterize the NPC1 compartment and NPC1 vesicle traffic by several independent methods, and the effects of NPC1 reduction or loss-of function on A. phagocytophilum cholesterol uptake and infection. Specific aim 2 is to determine the mechanism by which NPC1 vesicles traffic to Anaplasma inclusions. Our working hypothesis is that TGN-SNARE machinery is involved in transport of NPC1 vesicles containing LDL-CHOL to Anaplasma inclusions, and therefore is required for infection. To test the working hypothesis, our approach is to determine the intracellular localization of TGN-SNARE complexes associated with NPC vesicle transport and tethering proteins, and their requirement for Anaplasma cholesterol uptake and infection. Our approach is innovative, because cholesterol dependency of bacteria has not been used as a basis for the development of interventions. With respect to expected outcomes, the work proposed will identify the critical site of diversion of LDL-CHOL vesicular traffic that can be blocked, resulting in inhibition of Anaplasma infection without harming host cells. Such results are expected to have an important positive impact because the identified components and pathways are highly likely to provide new targets for prophylactic and therapeutic intervention in addition to fundamentally advancing the field of intracellular cholesterol regulation that will help growing problems of abnormal cholesterol homeostasis in the U.S. PUBLIC HEALTH RELEVANCE: The application is important to public health because Anaplasma phagocytophilum is now recognized one of the most prevalent life-threatening tick-borne disease in North America. At this time, treatment and prevention strategies are limited. Our work is to characterize how this bacterium, and related bacteria, hijacks intracellular cholesterol trafficking to promote bacterial survival. The results from this study will reveal nove targets for treatment and prevention. Furthermore, what is learned will contribute to a broader understanding of how essential cholesterol is regulated inside mammalian cells. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will reduce the burden of human disability.